1. Field of the Invention
The present invention relates to devices and methods for detecting and analyzing nucleic acids, proteins, macromolecules and other haptens. In particular, the invention includes a biochip. The invention is further directed to devices which the biochip comprises. Methods for making and using biochips and the devices which compose the biochip are also subjects of the present invention.
2. Background of the Invention
Nucleic acid probe technology has application in detection of genetic mutations and related mechanisms, cancer screening, determining drug toxicity levels, detection of genetic disorders, detection of infectious disease and genetic fingerprinting. Nucleic acid-based probe methods offer several advantages over conventional microbiological or immunological methods for detection of organisms, as described by Nakamura and Bylund (J. Clinical Laboratory Analysis, 6, 73-83, 1992). Utilizing biochip or microarray technology, one can conduct massively parallel experiments in the areas of genetics and proteomics in applications as diverse as pharmacogenomics, gene expression, compound screening, toxicology, Single Nucleotide Polymorphism (SNPs) analysis, Short Tandem Repeats (STRs) and molecular diagnostics.
Methods to amplify the number of copies of the nucleic acid available for detection of the signal generated after hybridization of the nucleic acid probe have been utilized. A review of nucleic acid based detection methods and various amplification schemes such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription based amplification, cycling probe reaction, Q.beta. replicase, and strand displacement amplification may be found in M. J. Wolcott, Clinical Microbiology Reviews, 5, Oct. 1992, pp 370-386.
U.S. Pat. No. 5,175,270 describes an amplification reagent consisting of layers of nucleotide polymers containing double stranded and single stranded sections. Each section has an end which is capable of hybridizing with another molecule.
Probe or hybridization assays are often based on the attachment of an oligonucleotide probe to a surface in order to capture a target nucleic acid molecule (analyte) from a sample. The attachment of this probe to the surface may be through covalent bonds or through a variety of passive absorption mechanisms (e.g., hydrophobic or ionic interactions).
U.S. Pat. No. 5,279,955 describes an immobilization process which uses a heterofunctional cross-linker for a plastic support. The cross-linker consists of a central ring which is hydrophobic and interacts with the plastic, and a hydrophilic chain which terminates in a group capable of reacting with a nucleic acid. Covalent attachment is achieved through a succinyl-olivetol-N-hydroxysuccinimide.
U.S. Pat. No. 5,262,297 describes immobilization of a probe through copolymers which contain reactive carboxylic acid groups and an 8-50 atom spacer with two or more unsaturated groups within the spacer.
U.S. Pat. No. 5,034,428 describes an immobilization process for probes in which a monomer is joined onto a hydrophilic solid support which can be irradiated in an oxygen free atmosphere. This method provides for covalent attachment of the probe.
U.S. Pat. No. 4,806,546 also describes an immobilization process for an amide modified nylon. The method relies on an amidine linkage under anhydrous conditions in the presence of an alkylating agent.
Maskos and Southern, 20 Nucleic Acid Research 1679, 1992, describe a linker system for the attachment of a nucleic acid to a glass support. The linker system allows for the chemical synthesis of a strand of nucleic acids on the surface. The primary advantage of the linker is that it is stable to an ammonia treatment which is required in the synthesis of the polynucleotide. A hexaethylene glycol spacer is incorporated into the linker which attaches to the glass through a glycidoxypropyl silane which terminates in a primary hydroxy group. The silane is condensed onto silane groups on a solid support. Additional cross-linking may be obtained by introducing water so that the epoxide group is cleaved to a diol. An acidic solution facilitates this process. The length of the linker may be varied by changing the spacer to ethylene glycol, pentaethylene glycol, etc.
Nucleic acid probes that have hybridized to their target sequence are detected based on various methods that introduce a detectable chemiluminescent, fluorescent or other identifiable label into a nucleic acid probe. Several of these techniques, are described in U.S. Pat. Nos. 4,968,602, 4,818,680, 5,104,791, and 5,272,056, and International applications W091/00926 and GB2169403A.
U.S. Pat. No. 5,283,174 (Arnold et al.), describes the use of a chemiluminescent label with DNA probes. The label is composed of an acridinium ester and has a number of desirable properties. It is stable to hybridization conditions, light is emitted only upon exposure to an alkaline peroxide, the emission kinetics are rapid, and the label on the unhybridized probe can be destroyed without an impact on the signal generated by the hybridized probe.
U.S. Pat. No. 5,089,387 describes a diffraction assay for the detection of DNA hybridization. In this invention, a solid support, generally silicon or polysilicon, is coated with a DNA probe. These surfaces are required to inherently adhere the DNA probe to the surface. Once the surface is coated with the probe, the surface is selectively inactivated to provide a series of very strictly controlled reactive probe lines for the generation of the diffraction grating. The unreacted surface is required to be non-light disturbing. The diffraction grating is only generated upon the addition of the analyte to the surface. The angle of diffraction is a function of the wavelength of incident light and the density and spacing of the individual gratings on the surface. A single detector or a multiple detector array may be used to detect and measure the light from all desired orders of the diffracted light.
U.S. Pat. No. 6,060,237 describes a light reflecting assay for the detection of nucleic acid hybridization. In the invention, an optically active solid support capable of producing a thin film effect is coated with an amplifying probe reagent able to bind to the target nucleic acid and create an increase in mass change on the optically active surface without disrupting the thin film effect. The direct optical thin film detection methods of the invention are extremely sensitive to changes in mass at the surface of the optically active support.
Mixed phase systems have typically been used to perform hybridization assays. In mixed phase assays the hybridizations are performed on a solid phase such as nylon or nitrocellulose membranes. For example, the assays usually involve loading a membrane with a sample, denaturing the DNA or creating single stranded molecules, fixing the DNA or RNA to the membrane, and saturating the remaining membrane attachment sites with heterologous nucleic acids to prevent the probe reagent from adhering to the membrane in a non-specific manner. All of these steps must be carried out before performing the actual hybridization.
Recent development of new technologies for synthesizing or depositing nucleic acids on substrates at very high densities have allowed the miniaturization of nucleic acid arrays yielding increased experimental efficiency and information content. (Lockhart & Winzeler, Genomics, Gene Expression and DNA Arrays, Functional Genomics, 405:6788, June 2000, 827-35). Utilizing these Biochips or Microarrays, one can conduct massively parallel experiments in genomics and proteomics with applications as diverse as, pharmacogenomics, gene expression, compound screening, toxicology, Single Nucleotide Polymorphism (SNPs) analysis, Short Tandem Repeats (STRs) and molecular diagnostics. It would be desirable to create a simple, cost-effective device that would produce a low background with optically clean fluorescent detection, allow oligonucleotides to be easily coupled in an array format, and be easily adapted to an automated manufacturing process.